Christophe Rothan

Publications of Christophe Rothan

• Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

  Authors: Carine Guillet, Mourad A M Aboul-Soud, Aline Le Menn, Nicolas Viron, Anne Pribat, Vé Ronique, Daniel Just, Pierre Baldet, Patrick Rousselle, Martine Lemaire-Chamley, Christophe Rothan

  PLoS ONE. 05/2012; 7(5-e36795):e36795.

  The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. WeThe SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the b-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 59 SlPPC2 promoter deletions fused to LUC in fruits at the 8 th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 59 UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars. Citation: Guillet C, Aboul-Soud MAM, Le Menn A, Viron N, Pribat A, et al. (2012) Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development. PLoS ONE 7(5): e36795. doi:10.1371/journal.pone.0036795 Copyright: ß 2012 Guillet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Institut National de la Recherche Agronomique (INRA), by the Région Aquitaine, and by a grant from EUSOL European Integrated Project [FOOD-CT-2006-016214 to NV]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

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• Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development.

  Authors: Carine Guillet, Mourad A M Aboul-Soud, Aline Le Menn, Nicolas Viron, Anne Pribat, Véronique Germain, Daniel Just, Pierre Baldet, Patrick Rousselle, Martine Lemaire-Chamley, Christophe Rothan

  PloS one. 01/2012; 7(5):e36795.

  The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. WeThe SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5' SlPPC2 promoter deletions fused to LUC in fruits at the 8(th) day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5' UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

• Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato.

  Authors: Aline Voxeur, Louise Gilbert, Christophe Rihouey, Azeddine Driouich, Christophe Rothan, Pierre Baldet, Patrice Lerouge

  The Journal of biological chemistry. 01/2011; 286(10):8014-20.

  L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has beenL-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.

• SNP discovery and linkage map construction in cultivated tomato.

  Authors: Kenta Shirasawa, Sachiko Isobe, Hideki Hirakawa, Erika Asamizu, Hiroyuki Fukuoka, Daniel Just, Christophe Rothan, Shigemi Sasamoto, Tsunakazu Fujishiro, Yoshie Kishida, Mitsuyo Kohara, Hisano Tsuruoka, Tsuyuko Wada, Yasukazu Nakamura, Shusei Sato, Satoshi Tabata

  DNA research : an international journal for rapid publication of reports on genes and genomes. 11/2010; 17(6):381-91.

  Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. SingleFew intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between 'Micro-Tom' and either 'Ailsa Craig' or 'M82'. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/.

• Genotype-dependent response to carbon availability in growing tomato fruit.

  Authors: Marion Prudent, Nadia Bertin, Michel Génard, Stéphane Muños, Sophie Rolland, Virginie Garcia, Johann Petit, Pierre Baldet, Christophe Rothan, Mathilde Causse

  Plant, cell & environment. 03/2010; 33(7):1186-204.

  Tomato fruit growth and composition depend on both genotype and environment. This paper aims at studying how fruit phenotypic responses to changes in carbon availability can be influenced byTomato fruit growth and composition depend on both genotype and environment. This paper aims at studying how fruit phenotypic responses to changes in carbon availability can be influenced by genotype, and at identifying genotype-dependent and -independent changes in gene expression underlying variations in fruit growth and composition. We grew a parental line (Solanum lycopersicum) and an introgression line from Solanum chmielewskii harbouring quantitative trait loci for fresh weight and sugar content under two fruit loads (FL). Lowering FL increased fruit cell number and reduced fruit developmental period in both genotypes. In contrast, fruit cell size was increased only in the parental line. Modifications in gene expression were monitored using microarrays and RT-qPCR for a subset of genes. FL changes induced more deployments of regulation systems (transcriptional and post-transcriptional) than massive adjustments of whole primary metabolism. Interactions between genotype and FL occurred on 99 genes mainly linked to hormonal and stress responses, and on gene expression kinetics. Links between gene expression and fruit phenotype were found for aquaporin expression levels and fruit water content, and invertase expression levels and sugar content. In summary, the present data emphasized age- and genotype-dependent responses of tomato fruit to carbon availability, at phenotypic as well as gene expression level.

• Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato.

  Authors: Elodie Mathieu-Rivet, Frédéric Gévaudant, Adrien Sicard, Sophie Salar, Phuc Thi Do, Armand Mouras, Alisdair R Fernie, Yves Gibon, Christophe Rothan, Christian Chevalier, Michel Hernould

  The Plant journal : for cell and molecular biology. 03/2010; 62(5):727-41.

  Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo-reduplication in connection with cell expansion and final fruit size determination. Endo-reduplication is anTomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo-reduplication in connection with cell expansion and final fruit size determination. Endo-reduplication is an impairment of mitosis that originates from the selective degradation of M phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo-reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo-reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over-expressing fruits, endo-reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo-reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo-reduplication in growth induction during tomato fruit development.

• An integrative genomics approach for deciphering the complex interactions between ascorbate metabolism and fruit growth and composition in tomato.

  Authors: Virginie Garcia, Rebecca Stevens, Laurent Gil, Louise Gilbert, Noé Gest, Johann Petit, Mireille Faurobert, Mickael Maucourt, Catherine Deborde, Annick Moing [......] Capucine Massot, Hélène Gautier, Martine Lemaire-Chamley, Antoine de Daruvar, Dominique Rolin, Bjoern Usadel, Marc Lahaye, Mathilde Causse, Pierre Baldet, Christophe Rothan

  Comptes rendus biologies. 11/2009; 332(11):1007-21.

  Very few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and otherVery few reports have studied the interactions between ascorbate and fruit metabolism. In order to get insights into the complex relationships between ascorbate biosynthesis/recycling and other metabolic pathways in the fruit, we undertook a fruit systems biology approach. To this end, we have produced tomato transgenic lines altered in ascorbate content and redox ratio by RNAi-targeting several key enzymes involved in ascorbate biosynthesis (2 enzymes) and recycling (2 enzymes). In the VTC (ViTamin C) Fruit project, we then generated phenotypic and genomic (transcriptome, proteome, metabolome) data from wild type and mutant tomato fruit at two stages of fruit development, and developed or implemented statistical and bioinformatic tools as a web application (named VTC Tool box) necessary to store, analyse and integrate experimental data in tomato. By using Kohonen's self-organizing maps (SOMs) to cluster the biological data, pair-wise Pearson correlation analyses and simultaneous visualization of transcript/protein and metabolites (MapMan), this approach allowed us to uncover major relationships between ascorbate and other metabolic pathways.

• Flexible tools for gene expression and silencing in tomato.

  Authors: Ana I. Fernandez, Nicolas Viron, Moftah Alhagdow, Mansour Karimi, Matthew Jones, Ziva Amsellem, Adrien Sicard, Anna Czerednik, Gerco Angenent, Donald Grierson, Sean May, Graham Seymour, Yuval Eshed, Martine Lemaire-Chamley, Christophe Rothan, Pierre Hilson

  Plant physiology. 10/2009;

  As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collection, ease of cultivation and transformation, and enables the analysis of biological processes impossible toAs a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collection, ease of cultivation and transformation, and enables the analysis of biological processes impossible to investigate with other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato, and CRC from Arabidopsis thaliana. Corresponding transcriptional fusions were made with the GUS gene, a nuclear localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues were confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR clonase reactions. Efficient silencing of the endogenous phytoene desaturase (PDS) gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the CaMV 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new NASC service unit dedicated to Solanaceae resources.

• The GDP-D-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non cellulosic cell wall biosynthesis in tomato.

  Authors: Louise Gilbert, Moftah Alhagdow, Adriano Nunes-Nesi, Bernard Quemener, Fabienne Guillon, Brigitte Bouchet, Mireille Faurobert, Barbara Gouble, David Page, Virginie Garcia, Johann Petit, Rebecca Stevens, Mathilde Causse, Alisdair R Fernie, Marc Lahaye, Christophe Rothan, Pierre Baldet

  The Plant journal : for cell and molecular biology. 08/2009;

  Summary The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-D-mannose to GDP-L-galactose, is generally considered as a central enzyme of the major ascorbate biosynthesis pathway inSummary The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-D-mannose to GDP-L-galactose, is generally considered as a central enzyme of the major ascorbate biosynthesis pathway in higher plants but experimental evidence for its role in planta is lacking. By using transgenic tomato lines RNAi-silenced for GME, we confirm that GME indeed plays a key role in the regulation of ascorbate biosynthesis in plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell expansion. A further remarkable feature of the transgenic plants was their exacerbated fragility and loss of fruit firmness. Analysis of cell wall composition of leaves and growing fruit revealed that cell wall monosaccharide content was altered in the transgenic lines, especially this directly linked to GME activity such as mannose and galactose. In agreement with this, immunocytochemical analyses showed an increase in mannan labelling in stem and fruit walls and in rhamnogalacturonans in the stem alone. Results from MALDI-TOF fingerprinting of mannanase cleavage products of cell wall suggest the synthesis of specific mannan structures with modified degrees of substitution with acetate in the transgenic lines. When considered together, these findings indicate the intimate linkage of ascorbate and non-cellulosic cell wall polysaccharide biosynthesis in plants, a fact that helps to explain general commonalities in seemingly unrelated traits such as fruit firmness and ascorbate content.

• Gene and metabolite regulatory network analysis of early developing fruit tissues highlights new candidate genes for the control of tomato fruit composition and development.

  Authors: Fabien Mounet, Annick Moing, Virginie Garcia, Johann Petit, Michael Maucourt, Catherine Deborde, Stephane Bernillon, Gwenaelle Le Gall, Ian Colquhoun, Marianne Defernez, Jean-Luc Giraudel, Dominique Rolin, Christophe Rothan, Martine Lemaire-Chamley

  Plant physiology. 02/2009;

  Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum L.) fruit. To get insights into the networksVariations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum L.) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12 to 35 days post anthesis - DPA). Transcriptome and metabolome analyses were carried out respectively with tomato microarrays and analytical methods including proton NMR and LC-MS. Pair-wise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes, e.g. the correlations between glutamine, bZIP and MYB transcription factors. Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

• Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.

  Authors: Moftah Alhagdow, Fabien Mounet, Louise Gilbert, Adriano Nunes-Nesi, Virginie Garcia, Daniel Just, Johann Petit, Bertrand Beauvoit, Alisdair R Fernie, Christophe Rothan, Pierre Baldet

  Plant physiology. 01/2008; 145(4):1408-22.

  L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized theL-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P(35S):Slgalldh(RNAi) silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P(35S):Slgalldh(RNAi) plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P(35S):Slgalldh(RNAi) plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.

• Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations.

  Authors: Rebecca Stevens, Michel Buret, Philippe Duffé, Cécile Garchery, Pierre Baldet, Christophe Rothan, Mathilde Causse

  Plant physiology. 05/2007; 143(4):1943-53.

  Fresh fruit and vegetables are a major source of ascorbic acid (vitamin C), an important antioxidant for the human diet and also for plants. Ascorbic acid content in fruit exhibits a quantitativeFresh fruit and vegetables are a major source of ascorbic acid (vitamin C), an important antioxidant for the human diet and also for plants. Ascorbic acid content in fruit exhibits a quantitative inheritance. Quantitative trait loci (QTL) for ascorbic acid content have been mapped in three tomato populations derived from crosses between cultivated tomato varieties (Solanum lycopersicum accessions) and three related wild species or subspecies. The first population consists of a set of introgression lines derived from Solanum pennellii, each containing a unique fragment of the wild species genome. The second population is an advanced backcross population derived from a cross between a cultivated tomato and a Solanum habrochaites (formerly Lycopersicum hirsutum) accession. The third population is a recombinant inbred line population derived from the cross between a cherry tomato line and a large fruited line. Common regions controlling ascorbic acid content have been identified on chromosomes 2, 8, 9, 10, and 12. In general, the wild alleles increased ascorbic acid content, but some improvement could also be provided by S. lycopersicum. Most QTLs appeared relatively stable over years and in different environments. Mapping of candidate genes involved in the metabolism of ascorbic acid has revealed a few colocations between genes and QTLs, notably in the case of a monodehydroascorbate reductase gene and a QTL present in two of the populations on chromosome 9 (bin 9-D), and a previously mapped GDP-mannose epimerase and a QTL on chromosome 9 (bin 9-J).

• Natural and artificially induced genetic variability in crop and model plant species for plant systems biology.

  Authors: Christophe Rothan, Mathilde Causse

  EXS. 02/2007; 97:21-53.

  The sequencing of plant genomes which was completed a few years ago for Arabidopsis thaliana and Oryza sativa is currently underway for numerous crop plants of commercial value such as maize, poplar,The sequencing of plant genomes which was completed a few years ago for Arabidopsis thaliana and Oryza sativa is currently underway for numerous crop plants of commercial value such as maize, poplar, tomato grape or tobacco. In addition, hundreds of thousands of expressed sequence tags (ESTs) are publicly available that may well represent 40-60% of the genes present in plant genomes. Despite its importance for life sciences, genome information is only an initial step towards understanding gene function (functional genomics) and deciphering the complex relationships between individual genes in the framework of gene networks. In this chapter we introduce and discuss means of generating and identifying genetic diversity, i.e., means to genetically perturb a biological system and to subsequently analyse the systems response, e.g., the changes in plant morphology and chemical composition. Generating and identifying genetic diversity is in its own right a highly powerful resource of information and is established as an invaluable tool for systems biology.

• The expression of cell proliferation-related genes in early developing flowers is affected by a fruit load reduction in tomato plants.

  Authors: Pierre Baldet, Michel Hernould, Frédéric Laporte, Fabien Mounet, Daniel Just, Armand Mouras, Christian Chevalier, Christophe Rothan

  Journal of experimental botany. 02/2006; 57(4):961-70.

  Changes in photoassimilate partitioning between source and sink organs significantly affect fruit development and size. In this study, a comparison was made of tomato plants (Solanum lycopersicum L.)Changes in photoassimilate partitioning between source and sink organs significantly affect fruit development and size. In this study, a comparison was made of tomato plants (Solanum lycopersicum L.) grown under a low fruit load (one fruit per truss, L1 plants) and under a standard fruit load (five fruits per truss, L5 plants), at morphological, biochemical, and molecular levels. Fruit load reduction resulted in increased photoassimilate availability in the plant and in increased growth rates in all plant organs analysed (root, stem, leaf, flower, and fruit). Larger flower and fruit size in L1 plants were correlated with higher cell number in the pre-anthesis ovary. This was probably due to the acceleration of the flower growth rate since other flower developmental parameters (schedule and time-course) remained otherwise unaffected. Using RT-PCR, it was shown that the transcript levels of CYCB2;1 (cyclin) and CDKB2;1 (cyclin-dependent kinase), two mitosis-specific genes, strongly increased early in developing flower buds. Remarkably, the transcript abundance of CYCD3;1, a D-type cyclin potentially involved in cell cycle regulation in response to mitogenic signals, also increased by more than 5-fold at very early stages of L1 flower development. By contrast, transcripts from fw2.2, a putative negative regulator of cell division in tomato fruit, strongly decreased in developing flower bud, as confirmed by in situ hybridization studies. Taken together, these results suggest that changes in carbohydrate partitioning could control fruit size through the regulation of cell proliferation-related genes at very early stages of flower development.

• Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato.

  Authors: Martine Lemaire-Chamley, Johann Petit, Virginie Garcia, Daniel Just, Pierre Baldet, Véronique Germain, Mathilde Fagard, Mariam Mouassite, Catherine Cheniclet, Christophe Rothan

  Plant physiology. 11/2005; 139(2):750-69.

  The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, itThe cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

• A fruit-specific phospho enolpyruvate carboxylase is related to rapid growth of tomato fruit.

  Authors: Carine Guillet, Daniel Just, Nathalie Bénard, Agnès Destrac-Irvine, Pierre Baldet, Michel Hernould, Mathilde Causse, Philippe Raymond, Christophe Rothan

  Planta. 04/2002; 214(5):717-26.

  Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement ofMalic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.

• Isolation and characterization of six peach cDNAs encoding key proteins in organic acid metabolism and solute accumulation: involvement in regulating peach fruit acidity.

  Authors: Christelle Etienne, Annick Moing, Elisabeth Dirlewanger, Philippe Raymond, René Monet, Christophe Rothan

  Physiologia plantarum. 03/2002; 114(2):259-270.

  As in many other fleshy fruits, the predominant organic acids in ripe peach (Prunus persica (L.) Batsch) fruit are malic and citric acids. The accumulation of these metabolites in fruit flesh isAs in many other fleshy fruits, the predominant organic acids in ripe peach (Prunus persica (L.) Batsch) fruit are malic and citric acids. The accumulation of these metabolites in fruit flesh is regulated during fruit development. Six peach fruit-related genes implicated in organic acid metabolism (mitochondrial citrate synthase; cytosolic NAD-dependent malate dehydrogenase, and cytosolic NADP-dependent isocitrate dehydrogenase) and storage (vacuolar proton translocating pumps: one vacuolar H+-ATPase, and two vacuolar H+-pyrophosphatases) were cloned. Five of these peach genes were homologous to genes isolated from fruit in other fleshy fruit species. Phylogenetic and expression analyses suggested the existence of a particular vacuolar pyrophosphatase highly expressed in fruit. The sixth gene was the first cytosolic NAD-dependent malate dehydrogenase gene isolated from fruit. Gene expression was studied during the fruit development of two peach cultivars, a normal-acid (Fantasia) and a low-acid (Jalousia) cultivar. The overall expression patterns of the organic acid-related genes appeared strikingly similar for the two cultivars. The genes involved in organic acid metabolism showed a stronger expression in ripening fruit than during the earlier phases of development, but their expression patterns were not necessarily correlated with the changes in organic acid contents. The tonoplast proton pumps showed a biphasic expression pattern more consistent with the patterns of organic acid accumulation, and the tonoplast pyrophosphatases were more highly expressed in the fruit of the low-acid cultivar during the second rapid growth phase of the fruit.